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What is serial block-face imaging?
Understanding the complex 3D architecture of cells and tissues is essential for the investigation of biological structure-function relationships. That is why biologists rely on 3D imaging techniques, such as physical sectioning or optical serial imaging, to study cellular ultrastructure and morphology. Traditional microtomy, however, relies on the production and imaging of individual sample sections, which can be challenging and time consuming. Modern methods allow for in-situ microtomy, which reduces manual steps and artifacts while increasing image quality.
Serial block-face imaging (SBFI), performed in scanning electron microscopes (SEMs), is an increasingly popular technique for the high-resolution imaging of cellular ultrastructure. Using these instruments, scientists can study cellular structures at nanometers resolution across millimeters of volume. Thermo Fisher Scientific supplies a range of these large volume analysis instruments, allowing scientists to observe how cells and organelles respond to diseases or various experimental methods. When combined with cryo-electron microscopy, large volume analysis can directly observe the context in which molecular interactions function in the cellular ultrastructure.
3D electron microscopy
Traditional microscopy is a valuable tool for high-resolution 2D imaging, but the inherent disadvantage is that these images require expert interpretation to extrapolate 3D structures. For example, a single cross-section of a microtubule may appear to be a simple circle or an ellipse. The user must evaluate the 2D image and then decide which section of the tubule it represents. With modern 3D electron microscopy, the entire volume is captured through the stacking of sequential images and the true 3D structure of the specimen can be observed.
3D electron microscopy is a critical tool for a number of biological applications that are not possible with 2D methods. One such application is connectomics, or the study of neuron interaction. In connectomics, images need to be collected at high resolution to show neuronal detail, but the data must also be collected across large volumes to ensure enough neuron-to-neuron connections are captured.
3D electron microscopy is also valuable for quantitative assessments. For example, the presence of mitochondria in cardiac tissue is used to assess heart health. 3D EM allows for large volume imaging of many individual cells; analytical software is then used to selectively view the mitochondria from within the whole data set.
Finally, experiments that search for rare events, such as ribbon synapses in retinal cells, are streamlined with 3D EM because large volumes of tissue can be collected automatically and then reviewed manually post-collection.
The area fraction of mitochondria vs total tissue is an indicator of how much energy the heart needs, and thus allows scientists to assess disease state.
How does serial block-face electron microscopy work?
During serial block-face imaging, the electron beam is first used to scan the surface of a resin-embedded tissue sample, capturing a 2D image of the specimen.
This top surface is subsequently removed with an in-situ microtome. The thickness of each section is user defined but is typically greater than 15-20 µm. Once this section is discarded, it is gathered by a debris collection device.
An image of the fresh surface is then collected with the SEM. This process is repeated until the whole sample has been imaged; total sample height can range from tens to hundreds of micrometers or more. The serial stack of images is then processed using 3D rendering software.
The serial block-face imaging process can be optimized and refined to match specific user or sample needs and requirements, including localized regions of interest, multiple areas, or various imaging detectors.
Schematic showing the basic layout used during serial block face electron microscopy.
Photograph taken within the SEM, showing the diamond knife (left) and the block face (middle) ready for SEM imaging.
Serial block-face imaging solutions from Thermo Fisher Scientific
The Thermo Scientific Volumescope 2 and Apreo 2 SEMs offer a novel serial block-face imaging solution with excellent Z-resolution that combines multi-energy deconvolution scanning electron microscopy (MED-SEM) with in-situ sectioning. The automation and ease-of-use of these instruments increases your productivity regardless of your level of expertise, granting isotropic resolution for your large volume samples.
Until now, the axial resolution in serial block-face imaging was limited by the minimal section thickness that can be physically cut with an in-chamber microtome. However, through the addition of multi-energy deconvolution SEM, our novel large-volume-analysis method now enables imaging with truly isotropic 3D resolution. Mechanical and optical sectioning are combined; data is acquired from multiple layers of the sample through the application of different beam energies between physical cuts.
Additionally, optimized detectors and low-vacuum operation ensure high-quality imaging, even for challenging resin-embedded samples. Unique acquisition software offers a range of automation, from low-level setup and alignment to walkaway acquisition of a complete image series. This includes a complete workflow from initial setup to final results, providing software solutions for large volume analysis, light microscopy image overlay, reconstruction, visualization, and segmentation.
Prof. Salisbury from the Mayo Clinic discusses his over 45 years of microscopy experience to answer various biological questions including recent 3D investigations with serial block face imaging to look at structure in normal and disease states.
Mouse retina imaged with large volume analysis. Dimensions: 9.65 x 10 x 25 µm; 10 nm isotropic resolution in HiVac mode; 1.18/1.78/2.27 kV, 100 pA; 1 µs dwell time; 250 images.
Zebrafish embryo head imaged with large volume analysis. Dimensions: 350 x 350 x 82.9 µm. 42 x 42 nm pixels in LoVac mode, 2 kV, 100 pA, 3 µs dwell time, 829 images at 100 nm. Sample courtesy of Robbert Creton, Brown University.
Rat Brain: Volume 85 x 85 x 123 um; 2.7 kV, 400pA, 2 us dwell time, 15 nm x 15nm x40 nm, 2133 images in LoVac. Sample courtesy of Grahame Knott, EPFL Lausanne. Data segmentation and visualization by Thermo Scientific Amira Software.
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Prof. Salisbury from the Mayo Clinic discusses his over 45 years of microscopy experience to answer various biological questions including recent 3D investigations with serial block face imaging to look at structure in normal and disease states.
Mouse retina imaged with large volume analysis. Dimensions: 9.65 x 10 x 25 µm; 10 nm isotropic resolution in HiVac mode; 1.18/1.78/2.27 kV, 100 pA; 1 µs dwell time; 250 images.
Zebrafish embryo head imaged with large volume analysis. Dimensions: 350 x 350 x 82.9 µm. 42 x 42 nm pixels in LoVac mode, 2 kV, 100 pA, 3 µs dwell time, 829 images at 100 nm. Sample courtesy of Robbert Creton, Brown University.
Rat Brain: Volume 85 x 85 x 123 um; 2.7 kV, 400pA, 2 us dwell time, 15 nm x 15nm x40 nm, 2133 images in LoVac. Sample courtesy of Grahame Knott, EPFL Lausanne. Data segmentation and visualization by Thermo Scientific Amira Software.
How it works.
Documents
Pathology Research
Transmission electron microscopy (TEM) is used when the nature of the disease cannot be established via alternative methods. With nano-biological imaging, TEM provides accurate and reliable insight for certain pathologies.
Plant Biology Research
Fundamental plant biology research is enabled by cryo electron microscopy, which provides information on proteins (with single particle analysis), to their cellular context (with tomography), all the way up to the overall structure of the plant (large volume analysis).
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